FNIP1 abrogation promotes functional revascularization of ischemic skeletal muscle by driving macrophage recruitment

Ischaemia of the heart and limbs attributable to compromised blood supply is a major cause of mortality and morbidity. The mechanisms of functional angiogenesis remain poorly understood, however. Here we show that FNIP1 plays a critical role in controlling skeletal muscle functional angiogenesis, a process pivotal for muscle revascularization during ischemia. Muscle FNIP1 expression is down-regulated by exercise. Genetic overexpression of FNIP1 in myofiber causes limited angiogenesis in mice, whereas its myofiber-specific ablation markedly promotes the formation of functional blood vessels. Interestingly, the increased muscle angiogenesis is independent of AMPK but due to enhanced macrophage recruitment in FNIP1-depleted muscles. Mechanistically, myofiber FNIP1 deficiency induces PGC-1α to activate chemokine gene transcription, thereby driving macrophage recruitment and muscle angiogenesis program. Furthermore, in a mouse hindlimb ischemia model of peripheral artery disease, the loss of myofiber FNIP1 significantly improved the recovery of blood flow. Thus, these results reveal a pivotal role of FNIP1 as a negative regulator of functional angiogenesis in muscle, offering insight into potential therapeutic strategies for ischemic diseases.

Figures-1-4-conclusively demonstrates the role of FNIP1 in regulation of macrophage recruitment and muscle angiogenesis.The RNA-seq data are not accessible from the database until 2024-09-19.It is impossible to evaluate the raw data presented in Figures 1 & 2.
Figure 5-demonstrates FNIP1 mediated regulation of angiogenesis in muscles through PGC-1alpha-and eventual macrophage recruitment.However, the exact mechanism of FNIP1 mediated regulation of PGC-1alpha remains absent in both the Result and Discussion sections.The authors have not made any attempts at least to consider or speculate the involvement of hyperactive molecular chaperone Hsp90 in upregulation of PGC1alpha (even at the transcriptional level) or any other client proteins involved in angiogenesis.
Figure 6-male vs female mice been considered in this study?Figure 8-Does AMPK play a role in this signaling?
Reviewer #3 (Remarks to the Author): In this manuscript, the authors investigate the role of FNIP1 in skeletal muscle angiogenesis.They find the KO of FNIP1 has increased vascular density and this is rescued by PCG1alpha KO but not by AMPK KO.They then investigate the role of recruited macrophages in the process.It was already known that the FLCN KO has increased vascular density that is rescued by mating with PCG1alpha KO, but not my rapamycin (Hasumi et al, 2012).In that paper, angiogenic genes were analyzed and not vascular density, but the changes in the muscle color that were presented made it clear that there was more vascularization.The fact that the FNIP1 has a similar role to FLCN is novel, but also not that surprising.The real added insight from this current submission is that the mechanism is based on immune cell recruitment.
Though the role of immune cells downstream of FNIP1 is very interesting, there are significant problems with the experiments that must be rectified.

Major:
Figure 1a shows a reduction in mRNA and protein with exercise but this is done in whole tissue.Which cells are expressing FNIP1?Do all expressing cells downregulate FNIP1?I realize from the western blots that the available antibody is non-specific and cannot be used for immunostaining.This could however be evaluated by in situ hybridization or RNAScope.This would also help in interpreting the TgKO FNIP1 and the MKO mice phenotypes.
The age of mice is not consistently reported.The methods say "8-14 weeks", but then one experiment is suddenly with 4-week-old mice.The age should be reported in the legend for every figure.
The ALP staining adds little to manuscript and is not a traditional vascular marker.It should be removed, unless the authors can explain how it stains for something other than vessel density.
Quantification that are "per HFP" are not acceptable since it does not allow for comparison with other published work.All quantification needs to be converted to "per um2" or "per mm2".
Microsphere injection as performed is not a recognized method to measure vessel leakiness, especially considering the resolution of the current images and the lack of co-staining for an endothelial cell marker.Extravasation of microsphere from leaky vessels occurs within microns of the vessel.The traditional method is to inject microspheres through the tail vein and then flush the vasculature with either PBS or PGA to wash out the beads within 5-10 minutes.The tissue is then costained with endothelial cell marker.Beads that remain after flushing had extravasated.The current method would only identify massive leakage and would not detect more subtle changes in permeability.This is not sufficient to claim that there is no permeability issues with the vessels.
Similarly, Evans blue needs an endothelial cell co-stain to normalize the Evans blue to the vascular density.
There are not enough details for the microsphere method.How long where they allowed to circulate?I also believed the authors meant to write 0.1 um not 0.1mM, but that is not clear.
Pericytes need two markers to be identified.PDGFRB also marks smooth muscle cells.Furthermore, the quantification should be normalized to vessel density.Therefore, the stainings need to be repeated with an endothelial cell marker and a second pericyte marker.
In the methods, the authors indicate that control mice are littermates.The methods also says that WT mice were purchased from GemPharmaTech.In the paper, control mice are listed as WT, which would indicate that they are not at all littermates (but instead purchased).The only way mice can be from the same litter is if the controls are heterozygotes (and not wildtype).The authors need to name the control/experimental by genotype.Hets should be indicated as "FNIP1+/-" or just "FNIP1 Het" and not as WT.If the control are indeed not littermates but purchased mice, then the experiments need to be repeated.Skeletal muscle density varies too much by strain to simply purchased separate control animals.
In the same vein, it is somewhat difficult to figure out how all these mice can be obtained as littermates.Listing the genotypes of the parents that were used to produce the experimental mice in the methods (or supplemental methods).Example "Het, TgKO and KO mice were obtained by mating FNIP1 tg/+ FNIP +/-mice with FNIP -/-mice".
In supplemental figure 1A, FNIP1 dependent and independent gene expression changes are identified.How can FNIP dependency be measured with the current experimental setup?I believe that this should be myocyte-dependent and independent (and therefore is just a typo), but I left this in "major" comments in case that is not the case.
The first paper from the authors on the TgKO had a different list of processes identified in the FNIP1dependent GO analysis (Xiao et al, Plos Genetics, 2021).Angiogenesis did not show up then.The description in the legend for that paper and this one are identical.The original paper stated 12-week-old mice and this paper states 8 to 14 weeks.Can the authors explain why the GO analysis is completely different?
All the bulk RNA-Seq is performed with n=2 per group.The authors state that there are 39 million reads per sample.And while this can be analyzed, and would be acceptable if it was for the analysis of a difficult to obtain tissues (such as a human biopsy), there is very little justification for not doing three biological replicates when working with mouse tissues.
In the FNIP1KO AMPKa1a2 f/fMyh5Cre mice, the loss of AMPK normalizes VEGF164 levels but does not normalize vascular density.Only the 188 isoform and Vegfb remain high.These are relatively minor players in angiogenesis.Have the authors investigated factors from outside the VEGF family that could explain the increase in vascular density?
When analyzing the presence of macrophages by qPCR, the authors use only markers of M1 macrophages (iNOS,TNFa,IL1b,etc.).And yet the phenotype is one of angiogenesis.Given that the most novel part of this manuscript is that the effect of FNIP1 KO in myocytes is to affect macrophage recruitment, the relative abundance of different subtypes of macrophages should be analyzed (by FACS, immunostaining, in situ, single cell RNA-Seq.. there are many options).M2-like markers such as IL10, CCL4, Ym1, TGFβ should be included.
The authors do not list which serotype is used in the methods of the text.I eventually found it in the experimental schematics shown in Figure 4c and 4f.The authors should just refer to it as AAV9 consistently.Furthermore, what was the promoter for construct?How old were the mice at the time of injection?
In the data reported in figure 4f, the amount of angiogenesis that has occurred within 7 days of injection is almost as high as what was observed after 8 weeks.I am wondering if my interpretation of the protocol as shown in 4f is correct.The mice receive AAV9 injection and chlodronate at D0, and then every 2 days afterwards, chlodronate liposomes are reinjected, until the animals are sacrificed at D7.If this is not correct, the figure 4f should be improved.Could the authors propose how a reasoning for such a huge increase within 1 week?Injecting AAV9-CCL8 and AAV9-CXCL13 barely increase vascular density (when compared to the increase seen in the FNIP1-MKO).Furthermore, these results represent pure correlation.A increases vascular density, B increases vascular density.. therefore A must be using B to increase vascular density.This is faulty logic.Furthermore, these experiments were done with 4-week-old mice, which makes it a developmental model rather than an adult model.The rest of the work uses adult models.I would remove these experiments.They need to be redone with the correct stage of mouse, but even then, they don't really add much.

Itemized Responses to Reviewer #1
"Reviewer #1: What appears to be the driving question of the manuscript is the relationship between myocytes (and in particular myocyte derived FNIP1 folliculin interacting protein 1 and the capillary density in skeletal muscle.Though not entirely, the models largely focus on mycoyte derived knock-out and over-expression studies.I will provide suggestions to the authors in two ways.First I will comment from front to back (not necessarily prioritized)" We sincerely appreciate the Reviewer's critical and constructive review of our manuscript.Our itemized responses are as follows: "1) There are syntax errors even in the abstract." We thank the Reviewer for the careful review.We have corrected grammar mistakes throughout the manuscript.
"2) The introduction, paragraph 1 has two "issues."The pathophysiology of brain vs. muscle ischemia is so vastly different and brain ischemia is not likely to be treatable by angiogenesis.Exactly what is functional angiogenesis?" We have removed the brain ischemia, thanks.For the "functional angiogenesis", we intend to emphasize the formation of patent, nonleaky, and pericyte-covered blood vessels, we have explained the functional angiogenesis in the revised Introduction text (page 4, line 68-69).
"3) Finding a higher capillary density that is expected or shown in the comparitor groups is not by definition angiogenesis.Going to much of Figure 1.Is the data in Fig 1 particularly 1b and 1c due to differences in EC proliferation, or cell death, or both.Identification of the driving process would allow more detailed vs. a cursory investigation.The models are driven by the myocyte expression but what are the expression levels in other "key" cells?" We thank the Reviewer.We have performed Ki67 staining assays to determine the proliferation of endothelial cells (EC) in skeletal muscles from NTG and MCK-FNIP1 Tg mice.We found that myofiber FNIP1 overexpression had no significant impact upon EC proliferation (new Supplementary Fig. 1c).Interestingly, TUNEL staining analysis showed that overexpression of FNIP1 in myofiber leads to increased EC apoptosis in skeletal muscles from MCK-FNIP1 Tg compared to NTG mice (new Supplementary Fig. 1d).This indicates that overexpression of FNIP1 in myofiber may affect the EC cell niches.
Notably, the muscle creatine kinase promoter (kind gift of E.N. Olson, University of Texas Southwestern) was used to generate myofiber-specific Fnip1 transgenic mice (MCK-FNIP1 Tg mice).The MCK-Fnip1 transgene transcript was efficiently expressed in skeletal muscle, and we observed no change in Fnip1 mRNA levels in the ECs isolated from MCK-FNIP1 Tg mice compared to NTG mice (new Supplementary Fig. 1b).
The above new data have been added to the revised new Supplementary Fig. 1b-d (page 7,Methods,page 28,line 648;page 30,31,. "4) Page 7 132 VEGFB ---often called large B which is a completely different gene from VEGFa or VEGF(isoform length)b -with b being within the a gene family but little b which can only be distinguished from a with specific probes" We thank the Reviewer.There are two isoforms of VEGF-B (VEGF-B167 and VEGF-B186) due to alternative splicing.We have designed RT-PCR primers to assess the levels of transcripts specific to each of these two VEGF-B isoforms.We found that both mRNA levels of Vegfb167 and Vegfb186 were significantly up-regulated in the muscles of the FNIP1 KO mice but reduced in FNIP1 TgKO mice (revised Fig. 1f).Moreover, FNIP1 deficiency-mediated induction of Vegfb167 and Vegfb186 mRNA expression were significantly reduced in the absence of PGC-1α but not AMPKα1/α2 (revised Fig. 3d, Supplementary Fig. 6d).These new data have been added to the revised Fig.1f,Fig. 3d,Supplementary Fig 3e,Fig. 6d, and new Supplementary Fig. 8a.
"6) Page 8 to 9 --Lots of genes are differentially expressed by bulk RNA-seq ---would it not be ideal to know how differences in EC number potenitally contribute or even better isolate EC from the muscle to look at EC specific gene expression." We followed the Reviewer's suggestion, we have quantified ECs (CD31 + , CD45 -) using a fluorescence-activated cell sorting (FACS)-based method (Zhang J et al., Cell Metab. 2020;31(6):113631(6): -1153.e7, PMID: 32492393).e7, PMID: 32492393).We observed a significant increase in the number of ECs from muscles of FNIP1 MKO mice relative to their littermate controls (revised Supplementary Fig. 3g,h).This is in line with enhanced muscle angiogenesis in FNIP1 MKO mice, and supports a role of FNIP1 in myofiber may affect the EC cell niches.We have also conducted RT-PCR analysis in ECs isolated from muscles of FNIP1 MKO mice.We found that EC-specific marker gene expression (Pecam1, Nos3, Kdr, Fli1, Ets2 and Cd34) were not changed in ECs isolated from FNIP1 MKO muscles (revised Supplementary Fig. 3i).These results suggest that an increase in EC number contribute to the observed changes in gene expression by bulk RNA-seq.This information has been added as revised Supplementary Fig. 3g-i  We appreciate the Reviewer's point.We have replaced the original Figure 2H with new representative images and re-quantified the data (revised Fig. 2l,m).We confirmed a significantly higher blood flow to the skeletal muscles in FNIP1 MKO muscles compared to controls (WT, 555.4 ± 41.0 PU vs. FNIP1 MKO, 1020.1 ± 83.5 PU, p < 0.001; n = 7-8 mice per group).

"8) Did the authors look at myoglobin?"
We found that the induced expression of myoglobin protein paralleled the activation of angiogenesis in FNIP1 MKO muscles (revised Supplementary Fig. 3f).The new data have been added to the revised Supplementary Fig. 3f (page 10, line 215-217).
As suggested, we have also conducted studies to examine the possibility that FNIP1 regulates factors known to inhibit angiogenesis.While the expression of many antiangiogenic genes (Ppargc1b, Thbs1, Thbs2, Vash1, and Endo1) was not different in the FNIP1 KO compared with the control muscles, Pedf and Plasminogen mRNA levels were decreased in FNIP1 KO muscles (revised Supplementary Fig. 4a).Interestingly, total AMPKɑ1/ɑ2 loss reversed the Plasminogen, but not Pedf, expression in FNIP1 KO muscles (revised Supplementary Fig. 4a).Together, FNIP1 deficiency induced a net proangiogenic signal that promote muscle angiogenesis in mice.
"10) Lines 286-291 ---a single gene is sufficient or solely responsible." We agree with the reviewer.Our data suggest that activation of either Ccl8 or Cxcl13 is capable of activating the infiltration of macrophages in skeletal muscles.However, the Reviewer#3 think that those data are not conclusive, and suggest to remove them from the manuscript.We sincerely appreciate the Reviewer's critical and constructive review of our manuscript.

Itemized Responses to
"These findings are significant and provide additional functional information towards FNIP1.The authors provide convincing high-quality data with addition of appropriate statistical analysis to support their claims.This manuscript is worthy of publication in the Nature Communications." Thank you for these positive comments.).We agree with the Reviewer in that it would be interesting to test whether the chaperone function of Hsp90 regulates PGC-1α.We have conducted additional experiments to address this intriguing question.First, we found that Hsp90 protein levels were not altered in FNIP1 MKO muscle (new Supplementary Fig. 7a) and we did not find evidence to suggest that PGC-1α protein may be a client of Hsp90 based on a public database (https://www.picard.ch/downloads/Hsp90interactors.pdf).To further determine whether the chaperone function of Hsp90 is involved in the regulation of PGC-1α, we have inhibited Hsp90 (by 17DMAG treatment) in C2C12 myotubes.We think it is worth noting that PGC-1α protein levels are hardly detectable in C2C12 myotubes, and we thus measured the PGC-1α mRNA levels.We have found that Hsp90 inhibition had no effect on PGC-1α mRNA levels (new Supplementary Fig. 7b).The effect of Hsp90 siRNAs were also assessed, siRNA-mediated knockdown of Hsp90 failed to affect PGC-1α mRNA levels (new Supplementary Fig. 7c).Together, these results suggest that Hsp90 may not involve in the regulation of PGC-1α mRNA levels.However, we cannot exclude the possibility that other Hsp90 client proteins are involved in FNIP1-meidated regulation of muscle angiogenesis.Future studies will be necessary to explore this interesting FNIP1/Hsp90 mechanism in muscle angiogenesis.The above points have been added as new Supplementary Fig. 7a-c (page 17-18, line 391-397; Methods, page 34, line 794-800) and addressed in the revised Discussion (page 24, line 538-544).6-male vs female mice been considered in this study?"

"Figure
We have not found any major phenotypic differences in the muscle angiogenesis results with the male versus female mice.The data in original Figure 6 showed muscle angiogenesis in male WT, FNIP1 KO and Fnip1 -/-, PGC-1α f/f/MCK-Cre (DKO) mice.We have now provided muscle angiogenesis data generated from female WT, FNIP1 KO and DKO mice as revised Supplementary Fig. 6h,i, showing PGC-1α-dependent regulation of muscle angiogenesis in FNIP1 KO mice of both sexes.Thus, muscle angiogenesis compared with littermate controls, were similar in female compared with male for FNIP1 KO lines.This point has been clarified further in the Methods on page 27, line 616-617.We appreciate the Reviewer's point.We concluded that FNIP1-regulated muscle angiogenesis does not require AMPK based on the muscle angiogenesis phenotypes of our total AMPKα1/α2 loss genetic mouse lines.We have removed the AMPK in Figure 8 model to clarify the AMPK-independent regulation of muscle angiogenesis by FNIP1.

Itemized Responses to Reviewer #3
"Reviewer #3: In this manuscript, the authors investigate the role of FNIP1 in skeletal muscle angiogenesis.They find the KO of FNIP1 has increased vascular density and this is rescued by PCG1alpha KO but not by AMPK KO.They then investigate the role of recruited macrophages in the process.It was already known that the FLCN KO has increased vascular density that is rescued by mating with PCG1alpha KO, but not my rapamycin (Hasumi et al, 2012).In that paper, angiogenic genes were analyzed and not vascular density, but the changes in the muscle color that were presented made it clear that there was more vascularization.The fact that the FNIP1 has a similar role to FLCN is novel, but also not that surprising.The real added insight from this current submission is that the mechanism is based on immune cell recruitment.
Though the role of immune cells downstream of FNIP1 is very interesting, there are significant problems with the experiments that must be rectified." We sincerely appreciate the Reviewer's critical and constructive review of our manuscript.Our itemized responses are as follows: Major comments: 1. "Figure 1a shows a reduction in mRNA and protein with exercise but this is done in whole tissue.Which cells are expressing FNIP1?Do all expressing cells downregulate FNIP1?I realize from the western blots that the available antibody is non-specific and cannot be used for immunostaining.This could however be evaluated by in situ hybridization or RNAScope.This would also help in interpreting the TgKO FNIP1 and the MKO mice phenotypes." We appreciate the Reviewers' critical points.Following the Reviewer's suggestions, we have conducted in situ hybridization experiments to stain Fnip1 mRNA within skeletal muscles.Our data suggest that Fnip1 mRNA is expressed in both myofibers and capillaries (new Supplementary Fig. 1a), which is consistent with previous reports that Fnip1 is ubiquitously expressed (Baba M et al.. Proc Natl Acad Sci U S A. 2006;103(42):15552-15557, PMID: 17028174).Interestingly, expression of the Fnip1 mRNA was reduced in both myofibers and capillaries after an acute bout of exercise (new Supplementary Fig. 1a).Moreover, efficient deletion of Fnip1 in skeletal muscle by KO was also verified by in situ hybridization (new Supplementary Fig. 1e), and the MCK-Fnip1 transgene transcript was expressed in a myofiber-specific manner (new Supplementary Fig. 1e).These new data have been added to the new Supplementary Fig. 1a,e  We thank the reviewer.We used a wider age range of mice at the beginning of the study.We have not found any major phenotypic differences in the results with the impact of age, we have now stated the age of the mice in all the Figure legends.

3, "
The ALP staining adds little to manuscript and is not a traditional vascular marker.It should be removed, unless the authors can explain how it stains for something other than vessel density." Alkaline phosphatase (ALP) staining can be used as an alternative marker for tissue vasculature, though it's not commonly applied.We have removed the data of ALP staining as requested.
4, "Quantification that are "per HFP" are not acceptable since it does not allow for comparison with other published work.All quantification needs to be converted to "per um2" or "per mm2"." As suggested, we have re-analyzed (plotted the data per mm 2 ) those data and updated all the relevant Figures.

5, "
Microsphere injection as performed is not a recognized method to measure vessel leakiness, especially considering the resolution of the current images and the lack of co-staining for an endothelial cell marker.Extravasation of microsphere from leaky vessels occurs within microns of the vessel.The traditional method is to inject microspheres through the tail vein and then flush the vasculature with either PBS or PGA to wash out the beads within 5-10 minutes.The tissue is then costained with endothelial cell marker.Beads that remain after flushing had extravasated.The current method would only identify massive leakage and would not detect more subtle changes in permeability.This is not sufficient to claim that there is no permeability issues with the vessel." We thank for these complimentary and constructive comments.We apologize that the description was not clear in the manuscript.To clarify this point, we would like to respectfully remind that we performed microsphere injection experiment using fluorescent Microsphere (100 nm) mainly for the visualization of perfused microvasculature, but not for the measurement of vessel leakiness.We have performed CD31 co-staining in microsphere injection experiment (revised Fig. 1g, Fig. 2d, Fig. 3e, Fig. 6h and Supplementary Fig. 3j,k) as suggested.These data indicate that the blood vessels induced by FNIP1 deficiency are patent and capable of sustaining blood flow.For vessel leakiness, our Evans blue (EB) dye infiltration test suggests that blood vessels induced by FNIP1 abrogation did not leak macromolecules EB (revised Supplementary Fig. 2e, Fig. 3l).In addition, please also see our response to the Reviewer#1 point #5 above.We have added new results to suggest that FNIP1 deficiency may act through PGC-1α to induce VE-cadherin 5 gene expression to improve endothelial barrier function (new Supplementary Fig. 7d-g).It would seem likely that myofiber FNIP1/PGC-1α signaling drives a concordant activation in muscle angiogenesis and endothelial barrier function to induce the formation of functional blood vessels.However, the Reviewer has pointed out the caveats and limitations regarding our current methods.We agree with the reviewers, the current method would only identify massive leakage and would not detect Sun et al.Nature Communications Manuscript #NCOMMS-23-05415A 10 more subtle changes in permeability.We have changed the language in the revised text to more accurately reflect the important point you raised.This information has been added as revised Fig. 1g,Fig. 2d,Fig. 3e,Fig. 6h,Supplementary Fig. 3j,page 11,page 17,page 18, and addressed in the revised Discussion (page 23, line 526-532).

6, "Similarly, Evans blue needs an endothelial cell co-stain to normalize the Evans blue to the vascular density."
As suggested, we have added CD31 co-staining in our Evans blue (EB) dye infiltration test (revised Supplementary Fig. 2e, Fig. 3l) as suggested.We confirmed that blood vessels induced by FNIP1 abrogation did not leak macromolecules EB into myofibers.
7, "There are not enough details for the microsphere method.How long where they allowed to circulate?I also believed the authors meant to write 0.1 um not 0.1mM, but that is not clear." The Reviewer is correct that it is 0.1 µm not 0.1 mM.We apologize for this error, which has now been fixed (page 8, line 171; page 29, line 664).We followed the published protocol for the in vivo visualization of blood flow using fluorescent Microsphere (Johnson C et al. Circ Res. 2004;94(2):262-268, PMID: 14670843).We have provided more details in the Methods section, including perfusion time (page 29, line 660-669).Briefly, to visualize in vivo blood flow, mice were anesthetized and perfused with 5-10 mL heparinized saline (10 U/mL) injected through the left ventricle.To identify capillaries with the capacity to be perfused through the circulatory system, we infused (10 mL, 1 mL/min) fluorescent microsphere (0.1μm, F8801, Invitrogen) at a 1:20 dilution in heparinized saline for 10 min, followed by euthanasia and tissue collection.Longitudinal cryosections (10 μm) of frozen gastrocnemius were processed and subjected to confocal microscopy for the visualization of perfused microvasculature.
8, "Pericytes need two markers to be identified.PDGFRB also marks smooth muscle cells.Furthermore, the quantification should be normalized to vessel density.Therefore, the stainings need to be repeated with an endothelial cell marker and a second pericyte marker." We have added the CD31 and PDGFRβ co-staining in skeletal muscles of FNIP1 MKO mice (revised Fig. 2f) as suggested.There was a positive correlation between capillary (CD31) and pericyte (PDGFRβ) densities and pericyte coverage of endothelial cells was unchanged (revised Fig. 2f-h).Muscle sections were also stained for chondroitin sulfate proteoglycan 4 (CSPG4; also named as NG2), a second pericyte marker, and similar results were obtained when we conducted CD31 and NG2 co-staining (revised Fig. 2i-k).This supports that blood vessels induced by myofiber FNIP1 deletion were covered by pericytes.We have also added the CD31/ PDGFRβ and CD31/NG2 co-staining in skeletal muscles of FNIP1 TgKO, DKO and TKO mice (revised Fig. 1i,j;Fig. 3f,g;Fig. 6j,k;Fig. 7d,e).These new data have been added as revised Fig. 1i,j;Fig. 3f,g;Fig. 6j,k;Fig. 7d,e (page 9,page 11,page 13,page 17,.
10, "In the same vein, it is somewhat difficult to figure out how all these mice can be obtained as littermates.Listing the genotypes of the parents that were used to produce the experimental mice in the methods (or supplemental methods).Example "Het, TgKO and KO mice were obtained by mating FNIP1 tg/+ FNIP +/-mice with FNIP -/-mice"." We thank the Reviewer and this was addressed in the point #9 above.11, "In supplemental figure 1A, FNIP1 dependent and independent gene expression changes are identified.How can FNIP dependency be measured with the current experimental setup?I believe that this should be myocyte-dependent and independent (and therefore is just a typo), but I left this in Sun et al.Nature Communications Manuscript #NCOMMS-23-05415A 12 "major" comments in case that is not the case." Because Fnip1 was only expressed in myocyte but not in other non-myocyte in FNIP1 TgKO muscles, we agree with the reviewer and we have changed "FNIP1-dependent and independent" to "myocyte-dependent and independent" to be more accurate.Thanks.
12, "The first paper from the authors on the TgKO had a different list of processes identified in the FNIP1-dependent GO analysis (Xiao et al, Plos Genetics, 2021).Angiogenesis did not show up then.The description in the legend for that paper and this one are identical.The original paper stated 12-week-old mice and this paper states 8 to 14 weeks.Can the authors explain why the GO analysis is completely different?" We think the Reviewer has raised an important point and we did not present our data clearly.To be clear, these are the same mouse models as in Xiao et al, Plos Genetics, 2021.Total RNA isolated from the entire gastrocnemius muscle of 8-week-old male mice was used for RNA-seq.For the GO analysis in Xiao et al, PLoS Genet.2021;17(3):e1009488, PMID: 33780446, the regulated genes were uploaded into DAVID Bioinformatics Resources 6.8.The Functional Annotation analysis using the "cellular compartments" defined by Gene_Ontology, Angiogenesis did not show up because "Angiogenesis" term is not belonged to "cellular compartments" that is not revealed in the analysis.In our current study, the regulated pathways were reviewed using the "Biological Process_ Direct" term defined by Gene_Ontology.This analysis revealed that the primary regulated genes belong to the angiogenesis biological process (Fig. 1e).Indeed, we also found that several terms such as "oxidation-reduction process" and "lipid metabolic process" are significantly enriched in FNIP1-regulated gene dataset (Fig. 1e), which is consistent with our previous reports related to "cellular compartments" analysis (Xiao et al, PLoS Genet.2021;17(3):e1009488, PMID: 33780446).We think that our results have uncovered a previously unrecognized role of FNIP1 that coordinately regulates mitochondrial function muscle fiber type and muscle angiogenesis.We have modified the descriptions in the revised Figure legends of Fig. 1e,Fig. 2c,Fig. 3c,and Fig. 6g to clarify this point.
13, "All the bulk RNA-Seq is performed with n=2 per group.The authors state that there are 39 million reads per sample.And while this can be analyzed, and would be acceptable if it was for the analysis of a difficult to obtain tissues (such as a human biopsy), there is very little justification for not doing three biological replicates when working with mouse tissues." We agree with the reviewer that an increase in sample size will help in this study.Unfortunately, because of the grant budget at the beginning of the study (2017) and the many mice groups were involved in this study, we used two independent muscle samples from each group for RNA-Seq.We would like to respectfully remind that FNIP1/PGC-1α regulation of muscle angiogenesis, the findings resulted from the unbiased global RNA-seq analyses, was validated by the RT-PCR.In addition, the functional relevance of this FNIP1/PGC-1α regulation of muscle angiogenesis has also been validated.We have also added a sentence to the revised Methods to clarify this point (page 32, line 751-753).

Sun et al.
Nature Communications Manuscript #NCOMMS-23-05415A 13 14, "In the FNIP1KO AMPKa1a2 f/fMyh5Cre mice, the loss of AMPK normalizes VEGF164 levels but does not normalize vascular density.Only the 188 isoform and Vegfb remain high.These are relatively minor players in angiogenesis.Have the authors investigated factors from outside the VEGF family that could explain the increase in vascular density?" We thank the Reviewer's insightful point.We agree with the reviewer, as we have also added new gene expression data demonstrating that the expression of the Vegf genes was induced by FNIP1 deficiency but not affected by the chlodronate treatment (see Figure 1a below).As further supported by the results of our new results, we speculate that factors from outside the VEGF family could be involved the activation of angiogenesis by FNIP1 deficiency.Indeed, we have conducted further comparative analysis and found that the expression of many angiogenesis factors was induced by FNIP1 deficiency but not affected by the total loss of AMPKα1/α2 in muscles (see Figure 1b below).Particularly, the Tnfa and Nos2, which are induced in FNIP1 KO muscles, have been shown to regulate vascular niche and angiogenesis (Sainson RC et al. Blood. 2008;111(10):4997-5007, PMID: 18337563;Hongu T et al. Nat Cancer. 2022;3(4):486-504, PMID: 35469015;Vågesjö E et al., Circ Res. 2021;128(11):1694128(11): -1707. PMID: 33878889;. PMID: 33878889;De Palma M et al. Nat Rev Cancer. 2017;17(8):457-474, PMID: 28706266).RT-qPCR confirmed that both Tnfa and Nos2 gene expression were significantly induced in FNIP1 KO muscles but not affected by the total loss of AMPKα1/α2 (see Figure 1c below).However, gene expression validation studies revealed that FNIP1 deficiency-mediated increased Tnfa and Nos2 expression was reduced after clodronate treatment (see Figure 1d below).Moreover, we also confirmed the induction of Tnfa and Nos2 expression was abolished in FNIP1 TgKO muscles (see Figure 1e below, revised Supplementary Fig. 5b), and muscle-specific disruption of PGC-1α resulted in marked decrease in Tnfa and Nos2 expression in the muscles of FNIP1 KO mice (see Figure 1f below, revised Fig. 6c).Together, the strong correlation between Tnfa and Nos2 expression and muscle angiogenesis in our serious mouse models suggest that Tnfa and Nos2 may be involved in FNIP1-regulated muscle angiogenesis.We have not delineated this interesting, albeit complex, mechanism.It will be of interest to conduct future studies to more precisely define this macrophage-dependent angiogenesis mechanism in skeletal muscle.This information has been added as new Supplementary  and addressed in the revised Discussion (page 25, line 570-575).15, "When analyzing the presence of macrophages by qPCR, the authors use only markers of M1 macrophages (iNOS,TNFa,IL1b,etc.).And yet the phenotype is one of angiogenesis.Given that the most novel part of this manuscript is that the effect of FNIP1 KO in myocytes is to affect macrophage recruitment, the relative abundance of different subtypes of macrophages should be analyzed (by FACS, immunostaining, in situ, single cell RNA-Seq.. there  As suggested, we have conducted additional immunostaining to examine the relative abundance of different subtypes of macrophages.As predicted, the FNIP1 MKO muscle cryosections stained for the M1 macrophage marker CD80 or M2 macrophage marker CD206 showed an increase in both and M2 macrophage recruitment compared with the control muscles (revised Fig. 4c,d).We have also included the results of RT-qPCR analysis of more M2-like markers genes (Il10, Ccl4, Ym1/Chil3, Tgfb1-3) from FNIP1 MKO muscle samples.Consistent with the immunostaining results, induction of both M1 and M2 macrophages marker genes expression was observed in muscle from FNIP1 MKO mice (revised Fig. 4b).Ischemia led to greater induction of both M1 and M2 macrophages marker genes expression in muscles from FNIP1 MKO mice (revised Fig. 7b).These results were added as revised Fig. 4b-d  16, "The authors do not list which serotype is used in the methods of the text.I eventually found it in the experimental schematics shown in Figure 4c and 4f.The authors should just refer to it as AAV9 consistently.Furthermore, what was the promoter for construct?How old were the mice at the time of injection?" We apologize that the description of this part was not clear in the previous versions.We have provided more details with regard to AAV9 injection in the Methods section (page 29, line 676-682).In brief, we generated recombinant AAV9 to express the Cre recombinase or GFP under the control of the CAG promoter.AAV9s were subsequently generated using packaging plasmids pAAV-helper and pAAV9 together with pAAV-CAG-Cre or pAAV-CAG-GFP.AAV9s were diluted in 0.9% NaCl at 1×10 13 Vp/mL, and injected into muscles (30 μL per TA muscle) of 8-week-old mice.AAV9-GFP was used as a control.17, "In the data reported in figure 4f, the amount of angiogenesis that has occurred within 7 days of injection is almost as high as what was observed after 8 weeks.I am wondering if my interpretation of the protocol as shown in 4f is correct.The mice receive AAV9 injection and chlodronate at D0, and then every 2 days afterwards, chlodronate liposomes are reinjected, until the animals are sacrificed at D7.If this is not correct, the figure 4f should be improved.Could the authors propose how a reasoning for such a huge increase within 1 week?" The Reviewer is correct.Schematic illustrating the clodronate treatment in the presence of AAV9-Cre-mediated FNIP1 ablation in skeletal muscle in original Figure 4F.The mice receive AAV9 injection and chlodronate treatment at D0, and then chlodronate liposomes are reinjected once every two days until the animals are sacrificed at D7. Surprisingly, our manipulation of FNIP1 in skeletal muscle induced robust angiogenesis within 7 days.We are also very interested in this observation.Evidence is emerging that macrophages are crucial regulators of vascular niche and angiogenesis in multiple animal models (Willenborg S et al. Blood. 2012;120(3):613-625, PMID: 22577176;Hirose N et al. Cell Transplant. 2008;17 16 activation and EC behavior.Specifically, in a 3D tissue-engineered human blood vessel networks in vitro, one-day exposure to macrophages has been shown to enhance angiogenesis and increase vessel formation (Graney PL et al. Sci Adv. 2020;6(18):eaay6391, PMID: 32494664).In addition, macrophages have also been to physically interact with blood vessels and contribute to the fusion of sprouting vessels (Fantin A et al. Blood. 2010;116(5):829-840, PMID: 20404134;Hsu CW et al. PLoS One. 2015;10(7):e0131643, PMID: 26132702).We are interested in this, and we have performed additional experiments to explore the potential macrophage-mediated EC cell niche activation.Ki67 staining showed that AAV9-based FNIP1 ablation in Fnip1 f/f muscles leads to a dramatic induction of EC proliferation, and clodronate treatment resulted in marked reduction of EC proliferation in the muscles from Fnip1 f/f mice injected with AAV9-Cre (revised Supplemental Fig. 5h,i).Moreover, TUNEL staining analysis showed that FNIP1 deficiency-mediated decreased EC apoptosis was prevented in the presence of clodronate (revised Supplemental Fig. 5j,k).It is tempting to speculate that such macrophage-mediated EC cell niche activation and angiogenesis are very active and rapid in skeletal muscle.We believe that studies aimed at defining this macrophage-dependent muscle angiogenesis mechanism would be an important focus of study for the future.This information has been added as revised Supplementary Fig. 5h-k (page 15, line 322-329; Methods, page 28, line 648; page 30, line 691-694) and addressed in the revised Discussion (page 23, line 516-525).
18, "Injecting AAV9-CCL8 and AAV9-CXCL13 barely increase vascular density (when compared to the increase seen in the FNIP1-MKO).Furthermore, these results represent pure correlation.A increases vascular density, B increases vascular density.. therefore A must be using B to increase vascular density.This is faulty logic.Furthermore, these experiments were done with 4-week-old mice, which makes it a developmental model rather than an adult model.The rest of the work uses adult models.I would remove these experiments.They need to be redone with the correct stage of mouse, but even then, they don't really add much." We have removed those AAV9-CCL8 and AAV9-CXCL13 experiments as suggested.Thanks.

19, "
The authors use luciferase system to show that PGC1alpha can induce CXCL13.Why not do a ChIP assay.This would be a direct assay and not depend on finding the correct co-factors." Thank you for this query.Unfortunately, we were unable to assess PGC-1α chromatin immunoprecipitation (ChIP) status due to the relative lack of sensitivity of the PGC-1α antibody in house, which work for heart and FNIP1 MKO muscle western blot.

20, "
The authors results imply that macrophage derived VEGF drives the phenotype.What are the VEGF levels after the chlodronate liposome injection.This rescue could be very informative for what factors are being secreted by the macrophages that induce angiogenesis."to macrophage-derived VEGF.We have added new gene expression data demonstrating that the expression of the Vegf genes was induced by FNIP1 deficiency but not affected by the chlodronate treatment (new Supplementary Fig. 8a).Please also see our response to the point #14 above.As supported by the results of our new results, we speculate that factors from outside the VEGF family could be involved in the activation of angiogenesis by FNIP1 deficiency.We have not fully delineated this interesting, albeit complex, mechanism.It will be of interest to conduct future studies to more precisely define this macrophage-dependent angiogenesis mechanism in skeletal muscle.This information has been added as new Supplementary  and addressed in the revised Discussion (page 25, line 570-575).
21, "Given that PGC1a KO rescues the phenotype, why are all the differentially regulated cytokines identified in 5C tested in the FNIP1KO PGC-1a f/fMCKCre mice?A different list of genes are tested.This could be very informative to identify which factor secreted by the myocytes is needed to recruit immune cells." We have now added new data demonstrating that Cxcl13 mRNA levels increase in FNIP1 KO muscles but are significantly suppressed in Fnip1 -/-, PGC-1α f/f/MCK-Cre muscles (revised Fig. 6d).All the differentially regulated cytokines identified in original Figure 5C are now tested in the Fnip1 -/-, PGC-1α f/f/MCK-Cre muscle.Many chemokine genes (e.g Ccl5, Ccl8, Cxcl9, Cxcl10, Cxcl13) are induced in FNIP1 KO muscles but suppressed in Fnip1 -/-, PGC-1α f/f/MCK-Cre muscles.One would predict that effective loss-of-function would be required targeting multiple of them.

Minor comments:
1. "The authors use the term "master regulator" on many occasions.That is a loaded term and should really be avoided.It implies that that factor can override all other factors, which rarely (or never) exists in biology." We have changed "master regulator" to "regulator" to be more accurate.Thanks.

"Spelling: agammaglobulinemic should be agammaglobulinemia."
We have corrected this typographical error.Thanks.

"On line 136, the authors refer to "fig 1g,f". It should be "1g,h"."
It should be Fig.1g,h instead of Fig. 1g,f.We apologize for this error, which has now been fixed.

"What are the units of figure 2i?"
The unit of Figure 2i is the Perfusion Unit (PU) for microcirculation blood perfusion.We thank the Methods,."7)Figure2-H cannot be used for what it is stating to measure.note the differences in signal in the tail." 4. "Figure 8-Does AMPK play a role in this signaling?"Sun et al.Nature Communications Manuscript #NCOMMS-23-05415A 7

Figure 1 .
Figure 1.Factors from outside the VEGF family could be involved in the activation of angiogenesis by FNIP1 deficiency.(a) Expression of genes (qRT-PCR) associated with angiogenesis in GC muscles from 8-week-old Fnip1 f/f mice injected with AAV9-Cre or control viruses following clodronate treatment.n = 4-7 mice per group.(b) Heatmap analysis of angiogenesis gene expression in skeletal muscle from indicated mice.(c, e, f) Expression of Tnfa and Nos2 (qRT-PCR) in GC muscles from the indicated mice.n = 4-5 mice per group.(d) Expression of Tnfa and Nos2 (qRT-PCR) in GC muscles from Fnip1 f/f mice injected with AAV9-Cre or control viruses following clodronate treatment.n = 4-7 mice per group.All data are shown as the mean ± SEM. *P < 0.05 vs. corresponding controls, #P < 0.05 versus AAV9-Cre, determined by one-way ANOVA coupled to Fisher's least significant difference (LSD) post-hoc test.
Reviewer #2 "Reviewer #2: In this manuscript by Sun et al, the authors attempted to decipher the mechanisms of functional angiogenesis in skeletal muscle.Skeletal muscle revascularization from ischemia is a very complex process involving interactions between several cell types and signals within the muscle microenvironment, including myofibers, endothelial cells and macrophages.The authors have shown that the co-chaperone FNIP1 expression in muscles is down regulated in response to exercise training.
the recovery of blood flow in the murine hindlimb ischemia model of peripheral artery disease.The authors concluded that FNIP1-macrophage signaling axis to control functional angiogenesis in skeletal muscle.These findings are significant and provide additional functional information towards FNIP1.The authors provide convincing high-quality data with addition of appropriate statistical analysis to support their claims.This manuscript is worthy of publication in the Nature Communications.However, there are few comments that the authors need to address." mediated regulation of PGC-1alpha remains absent in both the Result and Discussion sections.The authors have not made any attempts at least to consider or speculate the involvement of hyperactive molecular chaperone Hsp90 in upregulation of PGC1alpha (even at the transcriptional level) or any other client proteins involved in angiogenesis."Weappreciatethe Reviewer's insightful points.The exact mechanism of FNIP1-mediated regulation of PGC-1α in skeletal muscle remains unclear.It is surely a very important and intriguing question that warrants serious further characterizations.Previously published studies have revealed that FNIP1 could act as co-chaperone that regulates the chaperone function of Hsp90(Woodford et al., Nat Commun.2016;7:12037, PMID: 27353360; Sager et al., Cell Rep. 2019;26(5):134426(5): -1356.e5,PMID: 30699359;.e5,PMID: 30699359;  Sager et al., Trends Biochem Sci.2018;43(12):935-937, PMID: 30361061; Backe SJ et al., Cell Rep.  2022;40(2):111039, PMID: 35830801 -1-4-conclusively demonstrates the role of FNIP1 in regulation of macrophage recruitment and muscle angiogenesis.The RNA-seq data are not accessible from the database until 2024-09-19.It is impossible to evaluate the raw data presented in Figures1 & 2. (https://ngdc.cncb.ac.cn/gsa/s/22DHuXJ1).Thanks.2."Figure5-demonstrates FNIP1 mediated regulation of angiogenesis in muscles through PGC-1alpha-and eventual macrophage recruitment.However, the exact mechanism of FNIP1 are many options).M2-like markers such as IL10, CCL4, Ym1, TGFβ should be included." (1-2):211-222, PMID: 18468252; Juhas M et al.Nat Biomed Eng.2018;2(12):942-954, PMID: 30581652; Hongu T et al.Nat Cancer.2022;3(4):486-504, PMID: 35469015).It is worth noting that there is evidence that macrophages can rapidly impact vascular niche